Method for culturing cell and a culture vessel

ABSTRACT

A method for adhering and proliferating cell, which comprises the steps of inoculating, culturing and then killing fibroblast derived from a mammal, is provided. A culture vessel manufactured according to the steps of the method which can provide improved adhesion to cell and enhanced cell-proliferation is also provided.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to a method for adhering andproliferating cell. Particularly, the present invention relates to amethod for adhering and proliferating epithelial and hepatic cell. Thepresent invention also relates to a method for culturing epidermal cellto be used in an epidermal cell sheet and an epidermal cell suspensionwhich can be applied to an apellous part such as those with burn, wound,bedsore or skin ulcer for early reconstruction or treatment of suchdamaged tissue, an epidermal cell sheet and suspension prepared by theculture method, and a method for culturing hepatic cell which isimportant in analysis of hepatic function.

[0002] The present invention also relates to a culture vesselmanufactured according to the steps of the method of the presentinvention, which can provide improved adhesion to cell and enhancedcell-proliferation. Particularly, the present invention relates to aculture vessel which can provide improved adhesion to epithelial and/orhepatic cell and enhanced epithelial and/or hepatic cell-proliferation.More particularly, the present invention relates to a culture vesselthat enables preparation of an epidermal cell sheet and an epidermalcell suspension to be applied to an apellous part such as those withburn, wound, bedsore or skin ulcer for early reconstruction or treatmentof such damaged tissue.

[0003] Conventionally, two methods have widely been employed forculturing epithelial cell, particularly epidermal cell (or which iscalled epidermal keratinocyte). One utilizes sterilized 3T3 mouse lungfibroblast, i.e., viable 3T3 mouse lung fibroblast from which divisionand proliferation potencies have been deleted by irradiating, forexample, γ ray or by adding an agent such as mitomycin C, as feederlayer (such as the feeder layer culture method described in James G.Rheinwald and Howard Green. Cell 6: 331-344. Serial Cultivation ofStrains of Human Epidermal Keratinocytes: the Formation of KeratinizingColonies from Single Cells). The other utilizes serum-free medium suchas MCDB153 instead of feeder layer.

[0004] However, the conventional feeder layer culture method in which3T3 mouse lung fibroblasts are used as a feeder layer involvescomplicated procedure for preparing the 3T3 mouse lung fibroblastsimmediately before epidermal cells are inoculated, and such feeder layerhas a limited life time. During proliferation of epidermal cells otherthan mouse epidermal cells such as human epidermal cells and preparationof an epidermal cell sheet, those cells may possibly be contaminatedwith heterogenous cells, 3T3 mouse lung fibroblasts, and an agent suchas mitomycin C, which is added to delete division and proliferationpotencies of the 3T3 mouse lung fibroblasts, may remain.

[0005] On the other hand, when fibroblast homogenous to the epidermalcell and/or epidermal cell sheet of interest is used as the feeder cellinstead of 3T3 mouse lung fibroblast (e.g., human fibroblast may be usedfor preparing a human epidermal cell sheet), there is no possibility ofcontamination with heterogenous cells. However, their division andproliferation potencies should also be deleted by adding an agent suchas mitomycin C that may possibly remain. Human fibroblast may be used,but they provide slower proliferation rate of the epidermal cells whencompared to that obtained by using 3T3 mouse lung fibroblast.

[0006] Culture method employing serum-free medium may often provideslower proliferation rate of epidermal cell and require longer period oftime for incubation when compared to feeder layer culture methods whichemploy feeder cells such as 3T3 mouse lung fibroblast. Further,serum-free medium is likely to suppress the differentiation of epidermalcell, which may cause inability of the epidermal cell to form amultiple-layer, resulting in unsuccessful preparation of an epidermalcell sheet.

[0007] Japanese Unexamined Patent Application Publication No.285781/1987 disclosed a method which employs a feeder layer to culturehepatic cells. This method may also have a possibility of residual agentor contamination with heterogenous cells. Further, this method requirescontinual subculture of feeder cell to keep the cell ready for use underan optimal condition (subconfluent), making the procedure verycomplicated.

SUMMARY OF THE INVENTION

[0008] According to the investigation of various culture conditions forepidermal cell forming an epidermal cell sheet and hepatic cell, a novelmethod is found, which comprises the steps of inoculating, culturing andthen killing fibroblast derived from a mammal and separating the killedfibroblasts from the vessel at least partially to substantially leave acomponent or components, on the surface of the culture vessel, such asthe accumulated extracellular matrix which has been secreted from theculture cells. Thus, this method does not require preparing feeder cellssuch as fibroblasts nor sterilizing the cells as required inconventional feeder layer culture methods. Further, a culture vesselmanufactured according to the steps of the above method, which canprovide improved adhesion to cell and enhanced cell-proliferation, mayenable to be preserved for long time while keeping its property.Accordingly, the culture vessel does not require daily subculture (i.e.,transferring cells on another vessel in order to prevent overpopulationof the cells in the culture vessel) of fibroblasts to be used as thefeeder cells. Preservation of a large stock of the culture vessels canbe preserved in a cold and dark place. Further, the culture vessel whichcan provide improved adhesion to cell and enhanced cell-proliferationwhen compared to one manufactured by conventional methods can beobtained. On the basis of these findings, the present invention has beencompleted.

[0009] Accordingly, one object of the present invention is to provide amethod for adhering and proliferating cell comprising the steps ofinoculating, culturing and then killing fibroblast derived from amammal, which can provide improved cell adhesion and proliferationpotencies when compared to those provided by conventional feeder layerculture method, without preparing feeder cells such as fibroblasts norsterilizing such cells as required in conventional feeder layer culturemethods, and thus can provide an epidermal cell sheet, an epidermal cellsuspension or hepatic cells while avoiding contamination withheterogenous cells. Another object of the present invention is toprovide the followings: a method for culturing epidermal cell to be usedin an epidermal cell sheet and an epidermal cell suspension which can beapplied to an apellous part such as those with burn, wound, bedsore orskin ulcer for early reconstruction or treatment of the damaged tissue;an epidermal cell sheet and an epidermal cell suspension prepared by theculture method; and a method for culturing hepatic cells which isimportant in analysis of hepatic function.

[0010] Still another object of the present invention is to provide aculture vessel which can provide improved adhesion to cell and enhancedcell-proliferation according to the steps of the above method.Particularly, one object of the present invention is to provide aculture vessel which can provide improved adhesion to cell and enhancedcell-proliferation, which are manufactured by culturing and killingfibroblasts derived from a mammal (particularly 3T3 mouse lungfibroblast) by, for example, freezing and/or drying in a culture vesseland separating the killed fibroblasts from the vessel at least partiallyto substantially leave a component or components such as the accumulatedextracellular matrix which has been secreted from the culture cells toremain on the surface of the culture vessel, i.e., to leave component(s)necessary for cell adhesion and proliferation to remain on the surfaceof the culture vessel.

[0011] In summary, the present invention relates to a method foradhering and proliferating cell which comprises the steps ofinoculating, culturing and then killing fibroblast derived from amammal.

[0012] In the method, killed fibroblast may be preferably separated fromthe vessel partially, and more preferably entirely.

[0013] In the method, fibroblast may be killed by freezing, dryingand/or irradiating electromagnetic radiation or by repeating onetreatment selected from the group consisting of freezing, drying andirradiating electromagnetic radiation. Fibroblast may also be killed bya combination of at least two treatments selected from the groupconsisting of freezing, drying and irradiating electromagneticradiation. In the method, electromagnetic radiation may be at least oneselected from the group consisting of β ray, γ ray, X-ray, electron beamand UV ray.

[0014] In the method, 3T3 mouse lung fibroblast may be used asfibroblast derived from a mammal.

[0015] In the method, epithelial or hepatic cell may be used as the cellwhich adheres and proliferates. Epidermal cell is preferable asepithelial cell.

[0016] Further, the present invention relates to epidermal cell whichhas been cultured using the method. The present invention also relatesto an epidermal cell sheet or suspension prepared from the epidermalcell.

[0017] The present invention also relates to a culture vesselmanufactured according to the steps of the method, which can provideimproved adhesion to epithelial and/or hepatic cell and enhancedepithelial and/or hepatic cell-proliferation.

[0018] In the culture vessel, killed fibroblast may be preferablyseparated at least partially, or entirely.

[0019] Preferably, the culture vessel can be preserved by freezingand/or drying.

[0020] For the culture vessel, 3T3 mouse lung fibroblast is used asfibroblast derived from a mammal preferably.

[0021] For the culture vessel, epidermal keratinocyte may be usedpreferably as the epithelial cell.

[0022] The material of the culture vessel may be glass, syntheticpolymer or biopolymer. Further, the culture vessel may have any shapesuch as flask, petri dish, roller bottle, tray, well plate, beads, film,sheet or sponge.

[0023] The culture vessel made of glass or synthetic polymer may haveany shape such as flask, petri dish, roller bottle, tray, well plate,beads, film, sheet or sponge.

[0024] Preferably, the culture vessel made of biopolymer may have anyshape such as sheet, film, sponge or beads.

[0025] The culture vessel may be used to prepare an epidermal cellsheet.

[0026] The culture vessel may be used to prepare an epidermal cellsuspension.

BRIEF DESCRIPTION OF THE DRAWING

[0027]FIG. 1 illustrates a bar graph showing the average number ofepidermal cells collected from each of the culture flasks (culturesurface: 25 cm²) of frozen 3T3 and the control in Example 1 intriplicate by performing the test in triplicate.

DETAILED DESCRIPTION

[0028] Cells to adhere and proliferate in the cell adhesion andproliferation method according to the present invention include thosethat can be cultured by any conventional feeder layer culture method,such as epithelial cells and hepatic cell. The term “epithelial cell”herein encompasses the followings: epithelial cells such as mucosalepithelial cells (which are surface cells of intestine, oral cavity ornasal cavity) and corneal epithelial cells; and epidermal cells whichexist on skin epithelium and are denucleated and keratinized after celldivision. Hepatic cells are cells that constitute a hepatic lobule.

[0029] For the cell adhesion and proliferation method according to thepresent invention, it is important to kill fibroblast derived from amammal after culturing them separate from the vessel in order to removethe killed fibroblasts at least partially, and leave extracellularmatrix required for cell adhesion and proliferation on the surface ofthe culture vessel.

[0030] Fibroblasts may be used those derived from mammals such as mouse,human, rat, hamster and rabbit. Preferably, 3T3 mouse lung fibroblast,which is commonly used in conventional feeder layer culture methods, maybe used. The condition for inoculating and culturing cell is notparticularly limited, and any standard condition may be used. Forexample, fibroblasts grown in a culture vessel may be separated bytreating with trypsin solution (which was prepared by dissolving trypsin(0.25 weight/volume %) in a solution of 0.206 mg/mlethylenediamine-tetraacetic acid (EDTA) in phosphate buffer). Theseparated fibroblasts were suspended in a medium supplemented with 5 to10% fetal bovine serum, inoculated in the culture vessel, and then leftto stand in a CO₂ incubator. No special culture vessel, for example, aculture vessel coated with extracellular matrix such as collagen, isrequired. Any material or shape may be used for the culture vessel aslong as 3T3 fibroblasts, for example, can adhere to and proliferate inthe culture vessel. Any culture vessel for adhesive cell which arecommercially available such as flask, petri dish, roller bottle, wellplate or tray, or any carriers such as conventional synthetic polymermembrane, film or plate, or biopolymer membrane, film or microbeads maybe used, which can greatly reduce the process costs when compared to anyconventional methods.

[0031] An embodiment of culture vessel will be described which canprovide improved adhesion to cell and enhanced cell-proliferationaccording to the steps of the cell adhesion and proliferation methodwhich comprises the steps of inoculating, culturing and then killingfibroblast derived from a mammal.

[0032] Fibroblasts are grown in a culture vessel and the grownfibroblasts are killed. The killed fibroblasts (hereinafter referred toas “dead cells”) are then removed by separation, and component orcomponents required for cell adhesion and proliferation are left on thesurface of the culture vessel. As a result, property which can provideimproved adhesion to cell and enhanced cell-proliferation will berendered to the surface of the culture vessel. For the purpose ofrendering such property to the surface of the culture vessel,fibroblasts are inoculated preferably at 1.0×10² to 1.0×10⁵ cells/cm²,and more preferably at 3.0×10³ to 1.0×10⁵ cells/cm², and then culturedunder the above conditions.

[0033] In the subsequent step of killing the fibroblasts, it isimportant to kill substantially all the fibroblasts under the conditionwhich may not degenerate the component or components required for celladhesion and proliferation. Killing substantially all the fibroblastsmay facilitate and ensure the removal of the fibroblasts. Thus,contamination of the resultant epidermal cell sheet, epidermal cellsuspension or hepatic cells with heterogenous cells can be prevented.Further, unlike conventional sterilization such complete killing may notrequire adding an agent such as mitomycin C, thus avoiding possibilityof residual agent.

[0034] Fibroblasts may be killed by any method that may not degeneratethe component or components required for cell adhesion and proliferationnor inhibit the cell adhesion and proliferation. Such methods includefreezing, freeze-drying, drying, drying at low temperature, irradiationof electromagnetic radiation such as β ray, γ ray, X-ray, electron beamand UV ray, and a combination of such treatments. These methods canretain the property of the culture vessel which can provide improvedadhesion to cell and enhanced cell-proliferation. When epidermal cellsare inoculated and cultured in such a culture vessel, they may adhereand proliferate well to prepare an epidermal cell sheet for practicaluse.

[0035] Fibroblasts may be preferably killed by freezing and drying.Freezing may be easily manipulated, and can kill a large number offibroblasts at one time. Further, an apparatus to be used for freezingis generally cheaper than that used in other methods. After fibroblastsare killed, the culture device can be continually frozen for long-timepreservation while retaining its property which can provide improvedadhesion to cell and enhanced cell-proliferation. Freezing treatment maycomprise freezing and thawing, or repeated freeze-thawing procedure.Freezing treatment includes freeze-dryer, freezer, ultra-deep freezer,liquefied CO₂ or liquefied nitrogen gas. Any freezing temperature may beused which can freeze cells. Usually, 0° C. or lower may be used.Preferably, fibroblasts may be gradually frozen by using, for example, aprogrammable freezer since freezing rate may affect the death offibroblasts.

[0036] For drying treatment, any conventional dryer may be used. Dryingtemperature may be preferably from non-freezing temperature (around 0°C.) to 60° C., and more preferably 4° C. to 30° C. As described above,it is important to inhibit degeneration of active component(s).Therefore, fibroblasts may be desirably killed under these moderateconditions.

[0037] Fibroblasts may also be killed by irradiating electromagneticradiation selected from the group consisting of electron beam, γ-ray andUV-ray. Preferable range of irradiating electron beam or γ-ray is 5 to30 kGy, and preferable range of irradiating UV-ray is 50 to 5000mW·sec/cm². For the purpose of rendering property which can provideimproved adhesion to cell and enhanced cell-proliferation to the surfaceof the culture vessel, fibroblasts are inoculated preferably at 1.0×10²to 1.0×10⁵ cells/cm², and more preferably at 1.0×10³ to 1.0×10⁵cells/cm², and then cultured under the above conditions.

[0038] After culturing, culture supernatant of the fibroblast isremoved, and then the remainder is treated by irradiating 5 to 30 kGyelectron beams. Before irradiating electron beam or γray, i.e., afterculture supernatant of the fibroblast is removed, the culture vessel ispreferably kept at most 4° C. More preferably, a culture vessel istreated by irradiating ray after freezing treatment. Such conditions ofthe treatment prevent component or components required for cell adhesionand proliferation from degeneration. When a vessel is treated byirradiating electron beam keeping at 4° C., for example, adhesion tocell and cell-proliferation are a little inferior when compared tofeeder layer culture methods. However, an equivalent epidermal cellsheet can be prepared when compared to that obtained according to feederlayer culture methods. In the culture vessel which is treated byirradiating ray after freezing treatment, adhesion to cell andcell-proliferation are equal when compared to feeder layer culturemethods. Irradiating electron beam or γ-ray is useful to kill fibroblastcompletely and sterilize a culture vessel.

[0039] After fibroblasts are killed by the above treatment, dead cellsare removed by separation. The term “separate” or “separation” hereinintends to encompass all the manipulations for removing dead cells suchas detaching or washing.

[0040] In this process, fibroblasts should be completely removed inorder to prevent heterogenous cells such as 3T3 mouse lung fibroblastfrom contaminating the epidermal cell sheet, epidermal cell suspensionor hepatic cells obtained by using the culture vessel manufacturedaccording to the steps of the above cell adhesion and proliferationmethod, which comprises inoculating, culturing and then killingfibroblast derived from a mammal to provide improved cell adhesion andproliferation. However, complete removal of fibroblasts may not alwaysbe necessary in the aspect of cell adhesion and proliferation.Accordingly, the dead cells may be removed at least partially such thatcell adhesion or proliferation may not be inhibited. The term “beseparated at least partially” herein, which refers to the extent ofremoval of dead cells, particularly means removing preferably 50% ormore, more preferably 80% or more, and most preferably 100% of deadcells when compared to the total amount of viable fibroblasts justbefore killed.

[0041] Dead cells may be generally removed by rinsing the culturesurface with any isotonic solution which does not degenerate the activecomponent or components required for cell adhesion and proliferation,such as phosphate buffer, Hanks' solution and saline.

[0042] The extent to which dead cells are removed can be easilyconfirmed by using, for example, a phase contrast microscope. When 100%removal of dead cells is confirmed, then it means that the resultantepidermal cell sheet, epidermal cell suspension or hepatic cells arefree of heterogenous cells such as 3T3 mouse lung fibroblast. It is notdifficult to remove 100% of dead cells. Dead cells can be easily removedalmost completely by any conventional procedure. Alternatively, deadcells will completely be apart from the surface of the vessel into theculture solution. Therefore, an epidermal cell sheet, an epidermal cellsuspension or hepatic cells obtained by using the culture vesselaccording to the present invention which can provide improved adhesionto cell and enhanced cell-proliferation may be substantially free of anyheterogenous cell. Conventionally, a practical epidermal cell sheetcould be prepared only by feeder layer culture method which may causecontamination with heterogenous cells to some extent when cells derivedfrom a mammal other than human are used as the feeder cells. On theother hand, an epithelial cell sheet, i.e., a mucosal epithelial cellsheet or a mucosal epithelial cell suspension, or an epidermal cellsheet or an epidermal cell suspension obtained by using the culturevessel according to the present invention which can provide improvedadhesion to cell and enhanced cell-proliferation are free ofheterogenous cells.

[0043] The culture vessel which can provide improved adhesion to celland enhanced cell-proliferation (e.g., a petri dish containingcomponent(s) required for adhesion and proliferation of the target cellfrom the surface of which dead cells have been removed) can be left tostand, for example, in a refrigerator at 2° C. to 8° C. or in a deepfreezer at −30° C., −80° C. or the like for at least about 0.5 to 1 yearwhile keeping its property. This can eliminate several steps such aspreparing a feeder layer just before inoculating of epidermal cells,deleting division potency of, for example, 3T3 fibroblasts byirradiating γ-ray or by adding mitomycin C and thereafter inoculatingthe cells (1×10⁴ cells/cm²). Further, conventional feeder layers couldretain their property only for about 2 days. On the other hand, theculture vessel according to the present invention, which can provideimproved adhesion to cell and enhanced cell-proliferation, can bepreserved for about 0.5 to 1 year while retaining such property.Moreover, a large number of the culture vessel according to the presentinvention can be easily manufactured at one time, and they can providesimilar activity. Thus the process costs can be greatly reduced.Additionally, the method according to the present invention does notrequire daily subculture of fibroblasts for preparation of feeder layer.

[0044] The above epidermal cell sheet can be easily prepared in adesired amount when required, by using the culture vessel according tothe present invention. Particularly such an epidermal cell sheet may beprepared by a method similar to any conventional process for preparingepidermal cell sheet except for using the culture vessel according tothe present invention. For example, epidermal cells are inoculated(1.0×10⁴ cells/cm²) and then cultured for about 7 to 21 days whilechanging the medium about twice a week to prepare an epidermal cellsheet.

[0045] The above epidermal cell suspension can be prepared by anyconventional method for preparing-epidermal cell suspension except forusing the culture vessel according to the present invention. Forexample, epidermal cells are inoculated (1.0×10⁴ cells/cm²) and thencultured for about 3 to 21 days while changing the medium about twice aweek. Thereafter, the cells are treated with an enzyme such as trypsinto substantially obtain single cells which are then suspended in asolution such as neutral collagen solution.

[0046] Hereinafter, the present invention will be explained by way offollowing examples. These examples are not intended to limit the scopeof the present invention.

EXAMPLE Example 1

[0047] Established 3T3 mouse lung fibroblasts were inoculated in aculture flask (culture surface: 25 cm²) at 3×10³ cells/cm² and incubatedin a CO₂ incubator (at 37° C., 5% CO₂) for 4 days. The medium wasDulbecco's modified Eagle medium supplemented with 10% fetal bovineserum (DMEM+10% FBS).

[0048] After incubation, culture supernatant in the culture flask wasremoved by aspiration, and the remainder was left to stand in a deepfreezer at −85° C. for 12 hours for freezing. Next, the frozen culturewas thawed at room temperature, and the culture surface was rinsed with5 ml of phosphate buffer to remove dead cells. Then, the culture flaskwas again frozen by leaving it to stand in the deep freezer at −85° C.overnight.

[0049] The culture flask (hereinafter referred to as “3T3 frozen”) wasthawed at room temperature and inoculated with epidermal cells (1×10⁴cells/cm²) collected from human skin.

[0050] On the other hand, epidermal cells were inoculated at 1×10⁴cells/cm² in a culture flask (culture surface: 25 cm²) containing afeeder layer consisting of 3T3 mouse lung fibroblasts which have beentreated with mitomycin C to delete their division potency as a control.

[0051] These culture flasks were incubated in a CO₂ incubator (37° C.,5% CO₂) for 8 days. The medium was Green medium supplemented with 3%fetal bovine serum (Green+3% FBS).

[0052] After 8-day incubation, an epidermal cell sheet was prepared onthe surface of each culture vessel. The sheet was separated from theculture surface of each culture vessel by treating with dispase, theepidermal cells were treated with trypsin solution (which was preparedby dissolving trypsin (0.25 weight/volume %) in a solution of 0.206mg/ml ethylenediamine-tetraacetic acid (EDTA) in phosphate buffer) toobtain single cells, and the cells were counted on a hemacytometer.

[0053] The dispase treatment was performed by dissolving 10,000PUdispase in 10 ml of Dulbecco's modified Eagle medium to prepare dispasesolution, adding 3 ml of the dispase solution to the culture flask, andleaving the flask to stand in a CO₂ incubator for about 1 hour.

[0054] Epidermal cells were counted for both the control and 3T3 frozenin triplet, and the average of the results were shown in FIG. 1 forcomparison. As shown in FIG. 1, more epidermal cells were adhered andproliferated in the culture vessel according to the present invention(3T3 frozen) when compared to those grown by inoculating epidermal cellsaccording to the conventional feed layer culture method (control), whichemployed 3T3 mouse lung fibroblast as feeder cell, and incubating for 8days.

Example 2

[0055] Established 3T3 mouse lung fibroblasts were inoculated in aculture flask (culture surface: 25 cm²) at 3×10³ cells/cm² and incubatedin a CO₂ incubator (at 37° C., 5% CO₂) for 4 days. The medium wasDulbecco's modified Eagle medium supplemented with 10% fetal bovineserum (DMEM+10% FBS).

[0056] After incubation, culture supernatant in the culture flask wasremoved by aspiration, and the remainder was freeze-dried in a freezedryer. Schedule used was as follows: retaining at −30° C. for 1 hour;drying by vacuum aspiration; heating at 1.5° C./minute; and retaining at20° C. for 20 hours.

[0057] Epidermal cells collected from human skin were inoculated in theculture flask at 1×10⁴ cell/cm². The medium was Green mediumsupplemented with 3% fetal bovine serum. After 4 days, the colonies ofepidermal cells were observed. The epidermal cells were proliferatedwithout contamination with 3T3 mouse lung fibroblasts.

Example 3

[0058] Established 3T3 mouse lung fibroblasts were inoculated in aculture flask (culture surface: 25 cm²) at 3×10³ cells/cm² and incubatedin a CO₂ incubator (at 37° C., 5% CO₂) for 4 days. The medium wasDulbecco's modified Eagle medium supplemented with 10% fetal bovineserum (DMEM+10%FBS).

[0059] After incubation, culture supernatant in the culture flask wasremoved by aspiration, and the remainder was left to stand in a deepfreezer for 12 hours for freezing. Next, the frozen culture was thawedat room temperature, and the culture surface was rinsed with 5 ml ofphosphate buffer to remove dead cells. Then, the culture flask was againfrozen by leaving it to stand in the deep freezer at −85° C. overnight.

[0060] The culture flask (3T3 frozen) was thawed at room temperature andinoculated with epidermal cells collected from human skin at 1×10⁴cells/cm².

[0061] These culture flasks were incubated in a CO₂ incubator (37° C.,5% CO₂) for 8 days. The medium was Green medium supplemented with 3%fetal bovine serum (Green+3% FBS).

[0062] After 8-day incubation, epidermal cells were separated from theculture surface of each culture vessel, treated with trypsin solution(which was prepared by dissolving trypsin (0.25 weight/volume %) in asolution of 0.206 mg/ml ethylenediamine-tetraacetic acid (EDTA) inphosphate buffer) to obtain single cells, and then the cells werecounted on a hemacytometer. 3×10⁵ cells/ml epidermal cell suspension wasprepared by suspending cells in a neutral collagen solution(which wasprepared by dissolving aterocollagen derived from a hog (0.2weight/volume %) in Dulbecco's modified Eagle medium, and by adjustingat pH 7.4).

Example 4

[0063] Established 3T3 mouse lung fibroblasts were inoculated in aculture flask (culture surface: 80 cm²) at 3×10³ cells/cm² and incubatedin a CO₂ incubator (at 37° C., 5% CO₂) for 3 days. The medium wasDulbecco's modified Eagle medium supplemented with 10% fetal bovineserum (DMEM+10% FBS).

[0064] After incubation, culture supernatant in the culture flask wasremoved by aspiration, and then the remainder was treated by irradiating10kGy- and 25kGy- electron beam at 4° C.

[0065] Epidermal cells collected from human skin were inoculated in theculture flask at 1×10⁴ cell/cm². The medium was Green mediumsupplemented with 3% fetal bovine serum.

[0066] After that, epidermal cells were adhered and proliferated in theculture vessel. The epidermal cell sheet could be prepared. 3T3fibroblasts were observed to proliferate in the culture vessel which hadnot been treated by irradiating electron beam, but not observed in theculture vessel treated by irradiating electron beam.

What is claimed is:
 1. A method for adhering and proliferating cell,which comprises the steps of inoculating, culturing and then killingfibroblast derived from a mammal.
 2. The method according to claim 1,wherein said killed fibroblast is separated from the culture vessel atleast partially.
 3. The method according to claim 1, wherein said killedfibroblast is separated from the culture vessel entirely.
 4. The methodaccording to claim 1, wherein said fibroblast is killed by at least onetreatment selected from the group consisting of freezing, drying andirradiating electromagnetic radiation.
 5. The method according to claim4, wherein electromagnetic radiation is at least one selected from thegroup consisting of β ray, γ ray, X-ray, electron beam and UV ray. 6.The method according to claim 1, wherein said fibroblast is killed byrepeating one treatment selected from the group consisting of freezing,drying and irradiating electromagnetic radiation.
 7. The methodaccording to claim 6, wherein electromagnetic radiation is at least oneselected from the group consisting of β ray, γ ray, electron beam, UVray and X-ray.
 8. The method according to claim 1, wherein saidfibroblast is killed by a combination of at least two treatmentsselected from the group consisting of freezing, drying and irradiatingelectromagnetic radiation.
 9. The method according to claim 8, whereinelectromagnetic radiation is at least one selected from the groupconsisting of β ray, γ ray, electron beam, UV ray and X-ray.
 10. Themethod according to claim 1, wherein said fibroblast is 3T3 mouse lungfibroblast.
 11. The method according to claim 1, wherein said cell isepithelial cell.
 12. The method according to claim 11, in which saidepithelial cell is epidermal cell.
 13. The method according to claim 1,wherein said cell is hepatic cell.
 14. An epidermal cell sheet preparedfrom the epidermal cell which is cultured using the method according toclaim
 1. 15. An epidermal cell suspension prepared from the epidermalcell which is cultured using the method according to claim
 1. 16. Aculture vessel manufactured according to the steps of claim 1, which canprovide improved adhesion to at least one selected from the groupconsisting of epithelial and hepatic cells and enhancedcell-proliferation of at least one selected from the group consisting ofepithelial and hepatic cells.
 17. The culture vessel according to claim16, wherein said killed fibroblasts are separated from the vessel atleast partially.
 18. The culture vessel according to claim 16, whereinsaid killed fibroblasts are separated from the vessel entirely.
 19. Theculture vessel according to claim 16, which can be preserved by atreatment selected from the group consisting of freezing and drying. 20.The culture vessel according to claim 16, wherein said fibroblastderived from a mammal is 3T3 mouse lung fibroblast.
 21. The culturevessel according to claim 16, wherein said epithelial cell is epidermalcell.
 22. The culture vessel according to claim 16, which is made of amaterial selected from the group consisting of glass, synthetic polymerand biopolymer.
 23. The culture vessel according to claim 16, which hasa shape selected from the group consisting of flask, petri dish, rollerbottle, tray, well plate, beads, film, sheet and sponge.
 24. The culturevessel according to claim 22, which is made of glass and has a shapeselected from the group consisting of flask, petri dish, roller bottle,tray, well plate, beads, film, sheet and sponge.
 25. The culture vesselaccording to claim 22, which is made of synthetic polymer and has ashape selected from the group consisting of flask, petri dish, rollerbottle, tray, well plate, beads, film, sheet and sponge.
 26. The culturevessel according to claim 22, which is made of biopolymer and has ashape selected from the group consisting of sheet, film, sponge andbeads.
 27. The culture vessel according to claim 16, whereby anepidermal cell sheet can be prepared.
 28. The culture vessel accordingto claim 16, whereby an epidermal cell suspension can be prepared.